L-glutamine-zein-naringenin nanoparticles and applications thereof

Abstract

The present invention discloses a L-glutamine-zein-naringenin nanoparticle and its application, which is a nanoparticle formed by zein self-assembly encapsulating naringenin, indicating that electrostatic adsorption has chitosan oligosaccharide grafted with L-glutamine. The present invention is prepared by natural and non-toxic materials such as naringenin, zein, chitosan oligosaccharide, etc. The results show that the nanoparticle has been successfully synthesized, and at the same time, the drug encapsulation rate is high and the expectation of no toxicity to cells is achieved. Through in vitro cell experiments, it is confirmed that it has antioxidant activity, has pH responsiveness so that it can be specifically selected to be released in large quantities in the small intestine, and in addition, it can also improve cell insulin resistance and fat metabolism. The present invention uses nanoparticles constructed from natural plant materials, and the system is safe and non-toxic, can play the function of targeted intestinal transportation of hydrophobic active substances, improve drug absorption and utilization, and then produce therapeutic effects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to L-glutamine-zein-naringenin nanoparticles and applications thereof. Background Art

[0002] Diabetes has become a common chronic metabolic disease, of which type 2 diabetes mellitus (T2DM) accounts for more than 90%. It is currently believed that the pathogenesis of T2DM is the combination of insulin resistance and impaired pancreatic β-cell function leading to insufficient insulin secretion. Hyperglycemia can cause oxidative stress by triggering free radical production and damaging the endogenous redox defense system. Due to long-term metabolic disorders, a variety of complications are caused, mainly including liver damage, stroke, coronary heart disease, diabetic nephropathy, retinopathy, peripheral neuropathy, etc. Therefore, there is an urgent need to develop effective diabetes therapies, especially those that reduce the risk of serious complications.

[0003] The current clinical intervention methods for T2DM are mainly diet-exercise blood sugar control, subcutaneous insulin injection, and oral hypoglycemic drugs. Subcutaneous insulin injection is an invasive treatment method with poor patient compliance. Common oral hypoglycemic drugs currently include biguanides (metformin), sulfonylureas (gliclazide), thiazolidinediones (pioglitazone), α-glucosidase inhibitors (acarbose), dipeptidyl peptidase-4 inhibitors (sitagliptin) and glinilide (repaglinide). However, they have large side effects and single functions, and there is an urgent need to develop oral hypoglycemic drugs with fewer side effects and diverse functions.

[0004] There are various routes of drug administration in clinical practice. Compared with other methods, oral administration is the most commonly used, safer, and has better patient compliance. Compared with traditional oral drug delivery systems, nanocarriers can ensure that drugs are effectively delivered to the target site and can release drugs as needed. In addition, the preparation of nanocarriers with suitable ligands can improve the targeting of drugs and reduce toxicity. The properties of protecting the embedded drugs enhance the stability and solubility in the gastrointestinal tract. Oral nanomedicines can prolong the efficacy of drugs to reduce drug dosage and administration frequency.

[0005] In the past 30 years, natural compounds have played an important role in the development of new drug treatments; active substances of natural origin can be used to prevent or support the treatment of certain pathological conditions, including diabetes. Since artemisinin won the Nobel Prize in Physiology or Medicine, research on the treatment of various diseases with natural products has become increasingly popular. Compounds of natural origin have been favored by more and more researchers because of their many physiological activities in the treatment of diabetes and its complications; experiments have shown that some natural products have the function of improving blood sugar control without drugs or insulin injections, which is a very exciting discovery. Natural products used to treat type 2 diabetes mainly focus on the pathophysiological mechanism, which is the same as conventional drugs, such as regulating insulin secretion, alleviating insulin resistance, enhancing insulin sensitivity, inhibiting α-glucosidase activity, promoting incretin secretion, and inhibiting dipeptidyl peptidase IV activity.

[0006] In addition, compared with current mainstream drugs, natural products often have physiological activities such as improving fat metabolism, anti-oxidative stress, anti-inflammation and weight control, and can improve diabetic complications at the same time; in addition, natural products are generally non-toxic or low-toxic, mild in effect, with low risk of contraindications or harmful effects, and are suitable for long-term use. At present, natural health substances with the ability to treat diabetes are divided into the following categories: non-flavonoid polyphenols (such as resveratrol, curcumin, tannins and lignans), flavonoids (such as anthocyanins, epigallocatechin gallate, quercetin, naringenin, rutin and kaempferol), as well as quinones, phenylpropanoids, terpenes and natural polysaccharides and proteins.

[0007] Many pharmacological activities of naringenin have been reported in the scientific literature. Naringenin can reduce glucose absorption by the intestinal brush border, reduce glucose reabsorption by the kidneys, and increase glucose absorption and utilization by muscle and adipose tissue.

[14] In hepatocytes, naringenin treatment reduced triglyceride production and gluconeogenesis, thereby alleviating hyperglycemia and hyperlipidemia.

[15] Evidence also suggests that naringenin has a productive effect in the pancreas, rescuing the remaining beta cells and improving their ability to sense and respond to glucose. Overall, the health benefits of naringenin are wide-ranging, and the low toxicity of the molecule makes it a candidate for the treatment of insulin resistance and T2DM.

[0008] However, its poor biopharmaceutical and pharmacokinetic characteristics have largely limited the therapeutic effect of naringenin. Chitooligosaccharides have been considered as suitable nanocarriers for oral administration of naringenin, which can improve the properties of being easily hydrolyzed and degraded in the gastrointestinal environment, the compliance and pharmacokinetic properties of antidiabetic phytochemicals. Chitooligosaccharides are the only positively charged cationic basic amino oligosaccharides in nature, which have been shown to have hypoglycemic effects by increasing the insulin secretion capacity of pancreatic β cells, reducing hepatic gluconeogenesis, and enhancing the uptake of glucose by skeletal muscle.

[0009] Zein is a plant protein extracted from corn. It has low toxicity and good biocompatibility, self-assembly, degradability and resistance to gastric acid decomposition, and has been classified as a safe food additive (GRAS) by the U.S. Food and Drug Administration (FDA).

[0010] Over the past four decades, non-alcoholic fatty liver disease (NAFLD) has become one of the most common complications of type 2 diabetes. NAFLD is defined as the presence of hepatic fatty degeneration (determined by imaging or histological testing) and the absence of secondary causes of liver fat accumulation (i.e., excessive alcohol consumption, lipogenic drugs, or monogenic genetic diseases). Type 2 diabetic patients are prone to NAFLD due to abnormal metabolic system function, which leads to lipid metabolism disorders; and because type 2 diabetic patients have hepatic insulin resistance, this can easily promote a portion of NAFLD patients to develop non-alcoholic steatohepatitis (NASH), which in turn develops into severe cirrhosis and liver failure.

[0011] Abnormal glucose and lipid metabolism caused by type 2 diabetes can cause visceral ectopic fat accumulation and adipose tissue inflammation; in addition, patients' diet and intestinal flora imbalance can aggravate the progression of NAFLD. Visceral ectopic fat accumulation, which usually occurs in obese and hyperlipidemic type 2 diabetic patients, increases the degree of insulin resistance in the body and gradually induces liver necroinflammation (through Kupffer cell activation), while activating hepatic stellate cells and increasing the production of collagen matrix; NAFLD will gradually progress to advanced liver fibrosis, cirrhosis and even hepatocellular carcinoma in some cases. Poor diet (especially high fat and high fructose intake) and genetic factors (e.g., PNPLA3 polymorphism) in patients with type 2 diabetes may also play a role in the progression of NAFLD, increasing the risk of liver lipid accumulation and liver fibrosis; dietary changes may lead to intestinal flora imbalance, accompanied by the hepatotoxic effects of secondary bile acids. Intestinal microbial dysbiosis may alter the production of short-chain fatty acids (from the fermentation of dietary carbohydrates) and increase the production of lipopolysaccharide entering the portal circulation (lipopolysaccharide excreted by intestinal bacteria into the circulation due to increased intestinal permeability). This effect produces pro-inflammatory liver stimulation and increases the risk of NAFLD progression.

[0012] Naturally occurring biodegradable polymers are widely used in the field of drug delivery due to their rich physicochemical properties, biodegradability, high biocompatibility and low toxicity. Natural materials include protein-based polymers such as albumin, gelatin, zein, collagen and polysaccharides such as chitosan, agarose, dextran, hyaluronic acid, alginate, carrageenan and cyclodextrin. The advantages of using natural materials for drug delivery are the following points, such as easy surface modification, low toxicity and non-immunogenicity, and high biocompatibility with tissues and cells. Nanoparticle drug delivery systems based on natural biodegradable polymers have been shown to be a promising, feasible and safe option for controlled drug release. These nanoformulations are superior to macro-formulations or traditional formulations in terms of controlled release, targeted delivery and therapeutic effects. In addition, they can also help reduce the frequency of dosing while ensuring drug stability and protecting drug structure. In addition, by modifying or modifying natural materials, specific organs and tissues can be targeted, the bioavailability, solubility and permeability of potent drugs can be increased, and macromolecules such as proteins, hydrophobic drugs and genes can be delivered as oral drug carriers. With the in-depth research of a wide variety of natural biodegradable polymers, the development of safe and effective drug delivery systems has great research value. By rationally utilizing the physical and chemical properties of natural materials themselves and appropriately modifying or modifying them, drugs can be efficiently loaded and released, thereby improving the therapeutic effect of drugs. Summary of the invention

[0013] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide an L-glutamine-zein-naringenin nanoparticle and its application. The present invention successfully synthesized GCS-Zein-Nar nanoparticles loaded with naringenin through infrared spectroscopy detection results. The particle size and transmission electron microscopy detection results showed that the particle size of the nanoparticles was about 120nm and the morphology was approximately spherical. The encapsulation rate of GCS-Zein-Nar nanoparticles was tested to be as high as about 87%. The cell activity of HepG-2 cells treated with GCS-Zein-Nar nanoparticles for 12h and 24h was detected by CCK-8 method. The cell activity was high, which can indicate that the GCS-Zein-Nar nanoparticles are non-toxic. The antioxidant activity of GCS-Zein-Nar nanoparticles themselves and on cells was detected by DPPH method and cell flow cytometry, respectively, and the research showed that the GCS-Zein-Nar nanoparticles have good antioxidant properties. By simulating the gastrointestinal environment, it was proved that the GCS-Zein-Nar nanoparticles are pH-responsive and can be released in large quantities in the small intestine. In addition, the efficacy of GCS-Zein-Nar nanoparticles in improving insulin resistance and liver complications was tested by constructing insulin-resistant cells and fatty degeneration cells. The results showed that after nanomedicine treatment, the cells had a relatively obvious relief effect on insulin resistance, fat metabolism was improved, and the metabolic rate was accelerated.

[0014] In order to achieve the above object, the present invention is implemented by the following scheme:

[0015] By utilizing the self-assembly property of zein in an ethanol aqueous solution, naringenin was encapsulated to form negatively charged Zein-Nar nanoparticles; Glutamine was grafted onto chitosan oligosaccharides through an amide reaction to form GCS. By utilizing the charge difference between Zein-Nar nanoparticles and GCS, GCS was electrostatically adsorbed on the surface of Zein-Nar nanoparticles to form GCS-Zein-Nar nanoparticles. In the characterization study of GCS-Zein-Nar nanoparticles, the chemical composition of the nanoparticles was verified by infrared, the particle size and morphology were observed by transmission electron microscopy, the naringenin was verified to be coated with chitosan oligosaccharides based on potentiometric analysis, the encapsulation rate and drug loading of naringenin in GCS-Zein-Nar nanoparticles were obtained by detecting the absorbance value of naringenin in GCS-Zein-Nar nanoparticles, and the release rate of GCS-Zein-Nar nanoparticles was determined by simulating the gastrointestinal environment. In the in vitro study of GCS-Zein-Nar nanoparticles, the CCK-8 method was used to detect the toxicity of HepG-2 liver cancer cells 12h and 24h after GCS-Zein-Nar nanoparticles were treated, the DPPH method was used to detect the antioxidant activity of GCS-Zein-Nar nanoparticles, the flow cytometry experiment was performed on HepG-2 liver cancer cells before and after administration to detect the antioxidant activity of GCS-Zein-Nar nanoparticles on cells, and the glucose absorption of the constructed insulin-resistant cells after administration was detected to determine the efficacy of improving insulin resistance. For the study of the efficacy of type 2 diabetes complications, fat-degenerated cells induced by free fatty acids at a concentration of 1mM were selected to simulate the abnormal fat metabolism of type 2 diabetes patients, and cell lipid detection was performed after in vitro administration, and the data results were compared and analyzed to clarify its efficacy.

[0016] The first object of the present invention is to provide an L-glutamine-zein-naringenin nanoparticle.

[0017] The second object of the present invention is to provide a method for preparing L-glutamine-zein-naringenin nanoparticles.

[0018] The third object of the present invention is to provide L-glutamine-zein-naringenin nanoparticles prepared by the preparation method.

[0019] The fourth object of the present invention is to provide the use of L-glutamine-zein-naringenin nanoparticles in the preparation of antioxidant drugs.

[0020] The fifth object of the present invention is to provide the use of L-glutamine-zein-naringenin nanoparticles in the preparation of drugs for preventing and treating insulin resistance.

[0021] The sixth object of the present invention is to provide the use of L-glutamine-zein-naringenin nanoparticles in the preparation of a medicament for preventing and treating steatosis and / or fatty liver.

[0022] Therefore, the present invention claims to protect a kind of L-glutamine-zein-naringenin nanoparticles. The nanoparticles formed by the self-assembly of zein encapsulating naringenin show that chitosan oligosaccharides grafted with L-glutamine are electrostatically adsorbed.

[0023] Also claimed is a method for preparing L-glutamine-zein-naringenin nanoparticles, comprising the following steps:

[0024] L-glutamine is grafted onto chitosan oligosaccharide to obtain chitosan oligosaccharide grafted with L-glutamine (GCS), i.e., L-glutamine-modified chitosan oligosaccharide;

[0025] Zein self-assembled and gold encapsulated naringenin to obtain zein nanoparticles encapsulated with naringenin (Zein-Na Nar nanoparticles);

[0026] Chitosan oligosaccharide grafted with L-glutamine and zein nanoparticles encapsulating naringenin are fully reacted, electrostatically adsorbed, and purified to obtain L-glutamine-zein-naringenin nanoparticles (GCS-Zein-Nar nanoparticles).

[0027] Preferably, the specific method of L-glutamine grafting chitosan oligosaccharide is: after L-glutamine activates the carboxyl group, it fully reacts with L-glutamine to obtain chitosan oligosaccharide grafted with L-glutamine.

[0028] More preferably, the specific method of L-glutamine grafting chitosan oligosaccharide is: after L-glutamine activates the carboxyl group, the pH is adjusted to 5-7 and reacted for 6-36 hours, and then mixed with glutamine and fully reacted to obtain chitosan oligosaccharide grafted with L-glutamine.

[0029] More preferably, the pH is adjusted to 6.0 and the reaction is carried out for 12 hours.

[0030] More preferably, the sufficient reaction is: reacting at 25-80° C. for 0.5-24 hours.

[0031] Even more preferably, the sufficient reaction is: reacting at 60° C. for 24 hours.

[0032] More preferably, EDC and NHS are used to activate the carboxyl group of L-glutamine.

[0033] Even more preferably, the molar ratio of EDC and NHS to L-glutamine is 1-10:1-10:1.

[0034] Most preferably, the molar ratio of EDC and NHS to L-glutamine is 10:10:1.

[0035] More preferably, the molar ratio of L-glutamine to chitosan oligosaccharide is 1 to 3:1.

[0036] More preferably, the molar ratio of L-glutamine to chitosan oligosaccharide is 1:1.

[0037] More preferably, after sufficient reaction, the chitosan oligosaccharide grafted with L-glutamine is fully dialyzed in water.

[0038] Even more preferably, after sufficient reaction, the chitosan oligosaccharide grafted with L-glutamine is obtained by dialyzing in water for 2 days using a 500Da dialysis membrane.

[0039] Preferably, the specific method of zein self-assembly for gold encapsulation of naringenin is as follows: zein is fully dissolved and hydrated in a mixed solution of ethanol and water, fully mixed and reacted with naringenin, and purified to obtain zein nanoparticles encapsulating naringenin.

[0040] More preferably, the volume ratio of ethanol to water in the mixed solution of ethanol and water is 5-9:1-5.

[0041] More preferably, the volume ratio of ethanol to water in the mixed solution of ethanol and water is 4:1.

[0042] More preferably, the dissolution and hydration conditions are: stirring at 2 to 8° C. for 6 to 24 hours.

[0043] More preferably, the dissolution and hydration conditions are: stirring at 4° C. for 12 hours.

[0044] More preferably, the mass ratio of zein to naringenin is 10-20:1-5.

[0045] More preferably, the mass ratio of zein to naringenin is 4:1.

[0046] More preferably, the fully mixed reaction is stirred at 2 to 8° C. for 10 to 60 minutes.

[0047] More preferably, the fully mixed reaction is stirred at 4° C. for 30 min.

[0048] More preferably, the purification is performed by centrifugation at a speed of 3000 to 8000 rpm for 5 to 30 min to remove solids.

[0049] More preferably, the purification is performed by centrifugation at 5000 rpm for 10 min to remove solids.

[0050] Preferably, the mass ratio of the chitosan oligosaccharide grafted with L-glutamine to the zein nanoparticles encapsulating naringenin is 1-5:5-10.

[0051] More preferably, the mass ratio of the chitosan oligosaccharide grafted with L-glutamine to the zein nanoparticles encapsulating naringenin is 1:4.

[0052] Preferably, the purification is performed by centrifugation to remove large particle impurities and then freeze-drying the supernatant.

[0053] The L-glutamine-zein-naringenin nanoparticles prepared by the preparation method also fall within the protection scope of the present invention.

[0054] The present invention also claims the following:

[0055] Application of the L-glutamine-zein-naringenin nanoparticles in the preparation of antioxidant drugs.

[0056] Application of the L-glutamine-zein-naringenin nanoparticles in the preparation of drugs for preventing and treating insulin resistance.

[0057] Application of the L-glutamine-zein-naringenin nanoparticles in the preparation of a drug for preventing and treating fatty degeneration and / or fatty liver.

[0058] Preferably, the drug is a pH responsive drug.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] The present invention uses natural and non-toxic materials such as naringenin, zein, and chitosan oligosaccharide to prepare GCS-Zein-Nar nanoparticles. According to the external characterization data results of GCS-Zein-Nar nanoparticles, it is shown that GCS-Zein-Nar nanoparticles have been successfully synthesized, and at the same time, the drug encapsulation rate is high and the expectation of no toxicity to cells is achieved. Through in vitro cell experiments, it is confirmed that it has antioxidant activity and has pH responsiveness so that it can be specifically selected to be released in large quantities in the small intestine. In addition, it can also improve cell insulin resistance and fat metabolism. The present invention uses natural plant materials to construct GCS-Zein-Nar nanoparticles. The system is safe and non-toxic, can play the function of targeted intestinal transportation of hydrophobic active substances, improve drug absorption and utilization, and thus produce therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is the synthesis route of GCS-Zein-Nar nanoparticles.

[0062] Figure 2 This is the result of H NMR spectrum detection.

[0063] Figure 3 The results are from UV spectroscopy.

[0064] Figure 4 The particle size distribution of nanoparticles.

[0065] Figure 5 is the potential of the nanoparticles.

[0066] Figure 6 The morphology of nanoparticles under transmission electron microscopy.

[0067] Figure 7 is the encapsulation efficiency and drug loading of nanoparticles.

[0068] Figure 8 In vitro antioxidant activity of nanoparticles.

[0069] Fig. 9 The cytotoxicity of nanoparticles.

[0070] Fig.10 The effect of GCS-Zein-Nar nanoparticles on cellular glucose uptake.

[0071] Fig.11 This is the result of Oil Red staining.

[0072] Fig.12 The results are for the quantitative analysis of intracellular lipids.

[0073] Fig.13 is the in vitro release rate of naringenin from GCS-Zein-Nar nanoparticles. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0075] 1. Experimental reagents:

[0076] Zein, chitosan oligosaccharide, and naringenin were purchased from Shanghai MacLean Biochemical Co., Ltd., anhydrous ethanol and L-glutamine were purchased from Guangzhou Chemical Reagent Factory, and EDC / NHS was purchased from Shanghai Xishi Biotechnology Co., Ltd.

[0077] 2. Experimental instruments:

[0078] Sigma32184 high-speed refrigerated centrifuge, Japan HITACHI 7650 transmission electron microscope, Jiangsu Jintan Medical Instrument Factory 78-1 magnetic stirrer, Nikon microscope, British Malvern ZEN3600, Japan Olympus optical inverted microscope, Germany Bruker 400MHz nuclear magnetic resonance spectrometer.

[0079] Example 1 Synthesis of GCS-Zein-Nar Nanoparticles

[0080] Taking advantage of the self-assembly property of zein in an aqueous solution of ethanol, naringenin (Nar) is encapsulated to form negatively charged Zein-Nar nanoparticles; chitosan oligosaccharide (COS) is the only positively charged cationic alkaline amino oligosaccharide in nature. Glutamine is grafted onto chitosan oligosaccharide through an amide reaction to form L-glutamine-grafted chitosan oligosaccharide GCS. Taking advantage of the charge difference between Zein-Nar nanoparticles and GCS, GCS is electrostatically adsorbed on the surface of Zein-Nar nanoparticles to form GCS-Zein-Nar nanoparticles. The synthesis route of GCS-Zein-Nar nanoparticles is as follows: Figure 1 The specific synthesis method shown is as follows:

[0081] (1) Preparation of L-glutamine grafted chitosan oligosaccharide (L-glutamine modified chitosan oligosaccharide / chitosan oligosaccharide grafted with L-glutamine) GCS

[0082] Take 1g of chitosan oligosaccharide (COS, molecular weight 3000Da) and dissolve it in 70mL of deionized water; dissolve 0.3g of L-glutamine, 1.6g of EDC and 0.95g of NHS in 30mL of deionized water (the molar ratio of EDC and NHS to L-glutamine is 10:1) to obtain L-glutamine activation solution, adjust the pH of the L-glutamine activation solution to 6.0, and stir for pre-reaction at room temperature for 12h; then mix the above two solutions, the molar ratio of L-glutamine to chitosan oligosaccharide (calculated as monomer) is 1:1, and react at 60℃ for 24h;

[0083] After the reaction was completed, the product was dialyzed in deionized water for 2 days using a 500Da dialysis membrane, and the product was freeze-dried to obtain L-glutamine-grafted chitosan oligosaccharide (chitosan oligosaccharide grafted with L-glutamine) GCS.

[0084] (2) Preparation of GCS-Zein-Nar Nanoparticles

[0085] Prepare a 20 mg / mL zein ethanol solution (ethanol concentration 80% v / v), and stir overnight at 4°C to fully dissolve and hydrate the protein;

[0086] Take 1 mL of zein solution, add 5 mg of naringenin, place it at 4 ° C and stir for 30 minutes to fully mix it, and obtain a zein solution containing naringenin (in this process, zein self-assembles to encapsulate naringenin to form nanoparticles, namely Zein-Nar); dissolve L-glutamine-modified chitosan oligosaccharide in deionized water (1 mg / mL) to obtain a modified chitosan oligosaccharide solution; mix the zein solution containing naringenin with the L-glutamine-modified chitosan oligosaccharide solution in a volume ratio of 1:5 (the mass ratio of zein containing naringenin to L-glutamine-modified chitosan oligosaccharide is 4:1), stir for 10 minutes to form nanoparticles, and chitosan oligosaccharide is electrostatically adsorbed on the surface of Zein-Nar. Centrifuge at 5000 rpm for 10 minutes to remove large particle impurities, and retain the supernatant for freeze-drying to obtain GCS-Zein-Nar nanoparticles.

[0087] Example 2 GCS-Zein-Nar Nanoparticles H NMR Spectrum Detection

[0088] 1. Experimental Methods

[0089] 10 mg of the freeze-dried glutamine-grafted chitosan oligosaccharide prepared in Example 1 was dissolved in 0.6 mL of heavy water, and its hydrogen nuclear magnetic resonance spectrum was detected and compared with the hydrogen nuclear magnetic resonance spectra of glutamine and chitosan oligosaccharide alone.

[0090] 2. Experimental Results

[0091] The results of H NMR spectra are as follows Figure 2 As shown, nuclear magnetic resonance hydrogen spectrum detection can explain the structural composition and chemical groups of organic matter. Compared with COS, the characteristic peak of the amino group in glutamine appeared at about 5.4ppm in the spectrum of GCS, the double peak near 2.0ppm in the spectrum belongs to the methylene group in glutamine, and 3.8ppm belongs to the methine group in glutamine. The peaks near 4.0ppm and 3.2ppm in the spectrum belong to the unmodified amino group and methylene group on the chitosan oligosaccharide skeleton, indicating that glutamine has been successfully coupled to the chitosan skeleton.

[0092] Example 3 UV spectroscopy detection

[0093] 1. Experimental Methods

[0094] In order to detect whether L-glutamine is successfully grafted with chitosan, the molecular structure after the reaction is analyzed by nuclear magnetic resonance hydrogen spectrum. 10 mg of the freeze-dried L-glutamine-modified chitosan oligosaccharide (GCS), chitosan oligosaccharide and L-glutamine prepared in Example 1 are respectively weighed and dissolved in 0.7 mL of deuterated water, and the samples are divided into nuclear magnetic resonance tubes for detection; the freeze-dried glutamine-modified chitosan oligosaccharide (GCS), chitosan oligosaccharide and L-glutamine are prepared into 1 mg / mL aqueous solution, and the absorbance of the sample is detected in the wavelength range of 215 to 450 nm using an ultraviolet spectrometer.

[0095] 2. Experimental Results

[0096] UV spectrum test results are as follows Figure 3 As shown, the UV spectrum was used to verify the characteristic peak changes of the synthesized glutamine-grafted chitosan oligosaccharide. The UV spectrum showed the characteristic absorption peaks of glutamine, COS and GCS, among which the UV spectrum of GCS simultaneously showed the characteristic absorption peaks of glutamine at about 220nm and COS at about 280nm. This result verified the successful synthesis of glutamine-grafted chitosan oligosaccharide.

[0097] Example 4 Nanoparticle Size and Potential Characterization

[0098] 1. Experimental Methods

[0099] The GCS-Zein-Nar nanoparticles prepared in Example 1 were dissolved in 1% acetic acid solution and ultrasonicated for 15 minutes to fully disperse the GCS-Zein-Nar nanoparticles. 1 mL of the solution was added to the sample pool, and the particle size of the two nanoparticles (ZN, GZN) was determined by dynamic light diffraction (DLS). In order to detect the surface charge of the GCS-Zein-Nar nanoparticles, 200 μL of the GCS-Zein-Nar nanoparticle solution was added to the potential detection sample tank, and the Zeta potential of the nanoparticles was detected using a nanoparticle size potential instrument, and the data was exported.

[0100] 2. Experimental Results

[0101] The particle size distribution of nanoparticles is Figure 4 As shown, the particle size of the nanoparticles (Zein-Nar nanoparticles) prepared in Example 1 without chitosan oligosaccharide modification is about 60 nm, and the particle size of the nanoparticles (GCS-Zein-Nar nanoparticles) modified with GCS increases to about 110 nm, indicating that the thickness of the GCS layer is about 50 nm. The particle size is normally distributed, indicating that the surface of the GCS-Zein-Nar nanoparticles prepared in Example 1 is successfully modified with chitosan oligosaccharide.

[0102] The potential of the nanoparticles is Figure 5As shown, according to the potential analysis, the surface of the nanoparticles (Zein-Nar nanoparticles) without GCS modification is negatively charged, and the surface charge of the nanoparticles (GCS-Zein-Nar nanoparticles) modified with positively charged GCS is strongly reversed to positive charge. The negatively charged small intestinal epithelial cells can better absorb GCS-Zein-Nar nanoparticles through charge attraction.

[0103] Example 5 Transmission electron microscopy detection

[0104] 1. Experimental Methods

[0105] The suspension of GCS-Zein-Nar nanoparticles prepared in Example 1 was dropped onto a copper mesh covered with a carbon film and allowed to stand for 5 min. The excess water was then absorbed with filter paper and allowed to stand for 30 min until it was completely dry. A HITACHI 7650 transmission electron microscope was used with an electron accelerating voltage of 300 kV at 1× and 2×10 4 Observe the particle size and morphology at a magnification of 2.

[0106] 2. Experimental Results

[0107] The morphology of nanoparticles under transmission electron microscopy Figure 6 As shown, the nanoparticles (Zein-Nar nanoparticles) without GCS modification are round or oval in shape, with a diameter of about 70 nm, and the nanoparticles (GCS-Zein-Nar nanoparticles) modified with GCS are visibly coated with a layer of substance on the surface, and the diameter increases to about 120 nm, which once again confirms that the surface of the GCS-Zein-Nar nanoparticles prepared in Synthesis Example 1 is successfully modified with chitosan oligosaccharide.

[0108] Example 6 Encapsulation efficiency and drug loading of naringenin in nanoparticles

[0109] 1. Experimental Methods

[0110] The naringenin content in the GCS-Zein-Nar nanoparticles prepared in Example 1 was determined by spectrophotometry. 1 mg of naringenin was weighed for nanoparticle preparation, and the prepared nanoparticle dispersion was centrifuged at a speed of 12000 rpm for 30 min. After removing the supernatant, anhydrous ethanol was added to extract the naringenin in the nanoparticles. The maximum light absorption of the extract at 325 nm was measured by ultraviolet spectrophotometry. At the same time, naringenin was accurately weighed to prepare anhydrous ethanol solution (naringenin concentration was 10, 50, 100, 200, 300 mg / mL), and a standard curve was drawn after measuring the absorbance. The mass of naringenin in the nanoparticles was calculated according to the regression equation given by the standard curve. The encapsulation efficiency and drug loading were calculated according to the following formula:

[0111]

[0112]

[0113] Among them, M C To determine the content of naringenin in the obtained nanoparticles, M T is the dosage of naringenin in the preparation of nanoparticles, M N is the total mass of the nanoparticles.

[0114] 2. Experimental Results

[0115] Encapsulation efficiency and drug loading are important indicators for evaluating the performance of nanoparticles. Figure 7 As shown, the experimental results show that the naringenin encapsulation rate of Zein-Nar nanoparticles (ZN NPs) without GCS is about 81.97%, while the naringenin encapsulation rate of GCS-Zein-Nar nanoparticles (GZN NPs) containing GCS is about 88.25%. It can be found that the addition of GCS will increase the encapsulation rate of nanoparticles. It is speculated that the reason may be that Zein and GCS produce a certain degree of cross-linking through electrostatic interaction, resulting in a coating on the surface of the self-assembled Zein nanoparticles and a tighter internal structure, so the encapsulation rate is improved. In addition, the naringenin drug loading of Zein-Nar nanoparticles without GCS is about 73.05μg / mg, while the naringenin drug loading of GCS-Zein-Nar nanoparticles containing GCS is about 61.02μg / mg. The reason for the decrease in drug loading is that the addition of GCS increases the total mass of the nanoparticles, so the drug loading decreases.

[0116] Example 7 Characterization of the in vitro antioxidant activity of GCS-Zein-Nar nanoparticles

[0117] 1. Experimental Methods

[0118] The DPPH method was used to detect the in vitro antioxidant activity of the GCS-Zein-Nar nanoparticles and Zein-Nar nanoparticles prepared in Example 1; a 0.1 mM DPPH solution was prepared twice, each time 0.002 g of DPPH was dissolved in 50 mL of ethanol and stored in the dark. 100 uL of the sample solution + 100 μL of the DPPH alcohol solution (3 wells for each concentration) was measured to measure the absorbance at 517 nm, the average value was taken, the DPPH clearance rate of each concentration was calculated, and a line graph was made.

[0119] 2. Experimental Results

[0120] Since naringenin has a strong antioxidant effect, in order to detect whether the encapsulation of nanoparticles affects the antioxidant activity of naringenin, the DPPH method was selected to detect the antioxidant capacity of nanoparticles in vitro. The DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) method is an experimental method widely used to detect the antioxidant capacity of substances in vitro. DPPH is a single-electron free radical with an absorption peak at 517nm. As the free radical is removed, its absorbance will gradually decrease. The results are as follows Figure 8 As shown; it can be found that regardless of whether GCS is added or not, the nanoparticles show good free radical scavenging ability. The nanoparticles without GCS (Zein-Nar nanoparticles, ZN NPs) scavenged about 50% of the free radicals at a concentration of 500 μg / mL, while the nanoparticles containing GCS (GCS-Zein-Nar nanoparticles, GZN NPs) had a free radical scavenging rate of more than 50% at a concentration of 100 μg / mL. It can be found that the addition of GCS can improve the antioxidant activity of the nanoparticles. The reason is that GCS itself has a certain antioxidant activity. Therefore, the antioxidant ability of the GCS-Zein-Nar nanoparticles prepared in Example 1 is improved to a certain extent after the addition of GCS. In addition, the results prove that the antioxidant activity of naringenin itself is not destroyed after the encapsulation of the GCS-Zein-Nar nanoparticles prepared in Example 1, which also verifies from the side that the GCS-Zein-Nar nanoparticles prepared in Example 1 can effectively protect the biological activity of naringenin.

[0121] Example 8 GCS-Zein-Nar Nanoparticle Cytotoxicity Detection

[0122] 1. Experimental Methods

[0123] The human liver cancer cell line HepG2 cells used in the experiment were from Guangzhou Medical University. HepG2 cells were cultured in a DMEM high-glucose medium (containing 1% penicillin-streptomycin mixed double antibody) with a newborn calf serum concentration of 10% at 37°C and a 5% CO2 concentration. When the cells grew to about 80% in the culture flask, they were incubated at 37°C for 1 min using a 0.25% trypsin cell digestion solution. After the digestion was completed, 5 mL of complete culture solution was quickly added to terminate the digestion. The cells were fully blown down and centrifuged at 1000 rpm for 5 min. The upper culture medium was carefully removed from the centrifuged cells, and the complete culture medium was added to fully resuspend them. Finally, the cell suspension was added to a new culture flask (passaged at a ratio of 1:2-3) and continued to be cultured.

[0124] When the cells cover the bottom of the culture flask, take it out from the incubator and add 1 mL of 0.25% trypsin to digest it. When white fog appears at the bottom of the culture flask, add 4 mL of culture medium and pipette to suspend the cells.4 / well density was inoculated into a 96-well plate, and the cells were allowed to adhere to the wall and cultured for 12 hours. The cultured cells were taken out, the cell waste liquid was sucked off, PBS was added to wash twice, and fresh culture medium was added. GCS-Zein-Nar nanoparticles prepared in Example 1 with different concentration gradients (10, 50, 100, 200, 500 μg / mL) were added and incubated for 12 hours and 24 hours. After the incubation, the cell waste liquid was sucked off, PBS was added to wash twice, 100 μL of CCK-8 solution was added, and the absorbance at 490 nm was detected by an enzyme marker.

[0125] 2. Experimental Results

[0126] In order to verify whether the GCS-Zein-Nar nanoparticles prepared in Example 1 are toxic to cells, the CCK-8 method was used to detect the activity of HepG2 cells co-incubated with the nanoparticles. Fig. 9 As shown, the results show that the GCS-Zein-Nar nanoparticles prepared in Example 1 have good biocompatibility, and do not show toxicity to HepG2 cells during the incubation period. Moreover, as the incubation time increases, the GCS-Zein-Nar nanoparticles prepared in Example 1 still do not produce toxicity to the cells, and no significant decrease in cell activity was found compared with the control group. The cytotoxicity results show that the GCS-Zein-Nar nanoparticles prepared in Example 1 have good biocompatibility, no obvious toxicity in vitro, and have the potential for in vivo application.

[0127] Example 9 Effect of GCS-Zein-Nar Nanoparticles on Protecting against Insulin Resistance

[0128] 1. Experimental Methods

[0129] Take out the cultured cells from the incubator and add 5×10 4 / well density was inoculated into a 96-well plate, and the cells were cultured for 12 hours. The cell waste liquid was sucked off, PBS was added to wash twice, and a culture medium containing 25nM insulin was added to induce the cells to produce insulin resistance. The culture medium was replaced every 12 hours and cultured for 24 hours to stabilize the insulin resistance model. The above-mentioned insulin-resistant cell model was experimented, and a culture medium containing different concentrations of GCS-Zein-Nar nanoparticles (10, 25, 50, 100, 200 μg / mL) prepared in Example 1 was added to the insulin resistance model cells. After 24 hours of co-culture, fresh serum-free culture medium was added and cultured for 12 hours. After the culture was completed, the culture medium was removed and the remaining glucose content in the culture medium was detected using a glucose kit (glucose oxidase method).

[0130] 2. Experimental Results

[0131] In order to detect the effect of nanoparticles on alleviating insulin resistance, HepG2 cells were first induced to insulin resistance in a culture environment of 25nM insulin for 24 hours. After induction, the cells were treated with culture medium containing nanoparticles. After 24 hours of treatment, the changes in the cell's glucose uptake were detected. The experimental results are as follows Fig.10 As shown, after insulin-induced culture, the cell uptake of glucose decreased significantly, that is, insulin resistance. After incubation with the GCS-Zein-Nar prepared in Example 1 for 24 hours, it can be found that the cells have restored the uptake of glucose to a certain extent, that is, the insulin resistance of the cells has been alleviated to a certain extent. The GCS-Zein-Nar prepared in Example 1 shows a certain degree of therapeutic effect on insulin-resistant cells.

[0132] Example 10 Effect of GCS-Zein-Nar Nanoparticles on Protecting against Fatty Degeneration

[0133] 1. Experimental Methods

[0134] Take out the cultured cells from the incubator and add 5×10 4 / well density was inoculated into a 96-well plate, and the cells were cultured for 12 hours. The cultured cells were taken out, the cell waste liquid was sucked off, and PBS was added to wash twice, and a mixed culture medium containing 1mM free fatty acids and free fatty acids and different concentrations of GCS-Zein-Nar nanoparticles prepared in Example 1 (10, 25, 50, 100, 200 μg / mL) was added to induce fatty degeneration of the cells, and the culture medium was replaced every 12 hours. After 24 hours of co-culture, the cells were stained with oil red to observe the distribution of intracellular fat. After taking pictures, isopropanol was used for extraction, and the absorbance was detected at a wavelength of 490nm after removing the isopropanol to detect the relative content of total lipids in the cells.

[0135] 2. Experimental Results

[0136] In order to further test the therapeutic effect of nanoparticles on cell steatosis, the effects of different nanoparticle concentrations on cell steatosis were compared, and 1mM free fatty acids were selected to induce cells for 24 hours, and the changes in cell steatosis were analyzed after the treatment. Fig.11 and 12 As shown, compared with normal cells, the intracellular lipid staining of cells after free fatty acid induction culture was significantly increased; in the culture environment with the GCS-Zein-Nar nanoparticles prepared in Example 1, it was found that although the free fatty acids induced fatty degeneration of the cells, the fat content of the cells showed a downward trend with the increase of the nanoparticle concentration, which shows that the GCS-Zein-Nar nanoparticles prepared in Example 1 can reduce the fatty degeneration of the cells to a certain extent.

[0137] Example 11 Determination of the release rate of naringenin from GCS-Zein-Nar nanoparticles

[0138] 1. Experimental Methods

[0139] Prepare simulated gastric fluid (SGF) according to the formula of the United States Pharmacopoeia: dissolve 2.0g of sodium chloride, 3.2g of pepsin and 7.0mL of hydrochloric acid in ultrapure water, make up to 1000mL, and adjust the solution pH to 1.2±0.1 for use; prepare simulated intestinal fluid (SIF): dissolve 6.8g of potassium dihydrogen phosphate in 750mL of ultrapure water, add 77mL of 0.2mol / L sodium hydroxide solution, mix well, add 10g of pancreatic enzyme, make up to 1000mL, and adjust the solution pH to 6.8±0.1 for use.

[0140] The in vitro release of naringenin was studied in simulated gastric and intestinal fluid environments. 3 mL of the dispersion of GCS-Zein-Nar nanoparticles prepared in Example 1 was added to a dialysis bag (molecular weight cutoff 5000 Da), and then the dialysis bags were placed in 50 mL of simulated gastric and simulated intestinal fluids, respectively, and placed in a 37°C constant temperature oscillator for constant temperature release at a speed of 60 rpm. At predetermined time points (0.5, 1, 1.5, 2, 3, 4, 5, 6 h), 1 mL of digestive fluid was drawn from the simulated digestive fluid, and the simulated digestive fluid was replenished. The content of naringenin in the digestive fluid at different time points was measured using an ultraviolet spectrophotometer.

[0141] 2. Experimental Results

[0142] The results are as follows Fig.13 As shown, the gastric and intestinal environments were simulated respectively, and the release rate of the GCS-Zein-Nar nanoparticles prepared in Example 1 was determined by detecting the content of naringenin in the GCS-Zein-Nar nanoparticles prepared in Example 1 in the simulated gastric fluid and the simulated intestinal fluid. It was found that the GCS-Zein-Nar nanoparticles prepared in Example 1 were released in small amounts in the gastric environment and in large amounts in the intestinal environment.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A pH-responsive L-glutamine-zein-naringenin nanoparticle, characterized in that: Chitooligosaccharides grafted with L-glutamine are electrostatically adsorbed on the surface of nanoparticles formed by zein self-assembly encapsulating naringenin.

2. A method for preparing pH-responsive L-glutamine-zein-naringenin nanoparticles, characterized in that: The following steps are involved: L-glutamine is grafted onto chitosan oligosaccharide to obtain chitosan oligosaccharide grafted with L-glutamine; zein self-assembles to encapsulate naringenin to obtain zein nanoparticles encapsulating naringenin; chitosan oligosaccharide grafted with L-glutamine and zein nanoparticles encapsulating naringenin are fully reacted, subjected to electrostatic adsorption, and purified to obtain L-glutamine-zein-naringenin nanoparticles.

3. The preparation method according to claim 2, characterized in that: The specific method of L-glutamine grafting chitosan oligosaccharide is as follows: after the carboxyl group of chitosan oligosaccharide is activated, it is fully reacted with L-glutamine to obtain chitosan oligosaccharide grafted with L-glutamine.

4. The preparation method according to claim 2, characterized in that: The specific method of encapsulating naringenin by self-assembly of zein is as follows: zein is fully dissolved and hydrated in a mixed solution of ethanol and water, and is fully mixed and reacted with naringenin to obtain zein nanoparticles encapsulating naringenin.

5. The preparation method according to claim 2, characterized in that: The dosage ratio of chitosan oligosaccharide grafted with L-glutamine and zein nanoparticles encapsulating naringenin is 1-5:2-10.

6. L-glutamine-zein-naringenin nanoparticles prepared by the preparation method according to any one of claims 2 to 5.

7. Use of the L-glutamine-zein-naringenin nanoparticles according to claim 1 or 6 in the preparation of pH-responsive antioxidant drugs.

8. Use of the L-glutamine-zein-naringenin nanoparticles according to claim 1 or 6 in preparing a pH-responsive drug for preventing and treating insulin resistance.

9. Use of the L-glutamine-zein-naringenin nanoparticles according to claim 1 or 6 in the preparation of a pH-responsive drug for preventing and treating steatosis and / or fatty liver.

Images